Nitrogen-containing heterocyclic compound

ABSTRACT

As a result of an effort made by us for the purpose of developing a therapeutic agent having a bone formation-stimulating effect by promoting the functions of osteoblasts, the present inventors discovered that a certain nitrogen-containing heterocyclic compound exhibits a potent bone formation-stimulating effect on the osteoblast and thus can serve as an excellent prophylactic or therapeutic agent against a metabolic bone disease, whereby establishing the present invention. Thus, the present invention provides a 3,6-disubstituted 1,2,4-triazolo[4,3-b]pyridazine compound or a pharmaceutically acceptable salt thereof as well as a pharmaceutical composition comprising such a compound and a pharmaceutically acceptable carrier, especially a bone-forming agent.

TECHNICAL FIELD

The present invention relates to a nitrogen-containing heterocycliccompound having an ability of stimulating bone formation in anosteoblast or a pharmaceutically acceptable salt thereof.

BACKGROUND ART

A normal bone metabolism involves an equilibrium between the level ofbone resorption by osteoclasts and the level of bone formation byosteoblasts, by which a homeostasis is maintained. A metabolic bonedisease is considered to be developed once such a balance between thebone resorption and the bone formation is lost. This disease includesosteoporosis, osteitis fibrosa (hyperparathyroidism), osteomalacia andPaget's disease which affects the parameters of systemic bonemetabolism. The osteoporosis is observed frequently in postmenoposalwomen or old men, and causes a pain such as a lumbar pain and a bonefracture, and is seriously problematic especially in older patientswhose bone fracture readily leads to a systemic weakness and a dementia.For the purpose of treating or preventing such a bone disease, a calciumpreparation, active vitamin D₃ preparation, calcitonin preparation andestrogen preparation are employed.

However, most of these therapeutic agents do not exhibit marked boneformation-stimulating effect, although they were reported to have a boneresorption-inhibiting effect. A bone-forming agent is highly desiredespecially in a senile osteoporosis which was reported to be causedmainly by a reduction in the bone formation due to a reduction in thebone turnover (New Eng. J. Med. 314, P1676, (1986)).

Recently, a benzothiepin derivative (for example, JP-A-8-231569) and anN-quinolylanthranilic acid derivative (for example, JP-A-9-188665)having an alkaline phosphatase-inducing activity were reported to beuseful in promoting osteogenesis and in treating a metabolic bonedisease. Nevertheless, their clinical utility is unknown.

On the other hand, there are the following reports with regard totriazolopyridazine derivatives (symbols in the description represent thesymbols in Formula (I) of the invention described below). However, anyof these references and patent specifications does not contain anydisclosure or suggestion of an osteogenesis-promoting effect.

(1) An antibacterial compound wherein Ra and Rb are taken together withan adjacent N atom to form a piperidino, E is a single bond, and R ispiperidino disclosed in U.S. Pat. No. 3,957,766; a method forsynthesizing a compound wherein R is an unsubstituted phenyl disclosedin Tetrahedron, 22 (7), 2073-9 (1966); a structure of a compound whereinR is p-(trifluoromethyl)phenyl or p-chlorophenyl disclosed in CASRegistry File under the codes RN=289651-67-8 and 202820-26-6; and acompound wherein R is o-nitrophenyl disclosed in SPECS catalog underRefcode:AG-690/3073051.

(2) A triazolopyridazine derivative having a bronchodilating effectwhich is a compound wherein Ra and Rb are taken together with anadjacent N atom to form a 4-methyl-1-piperazinyl, E is a single bond,and R is an unsubstituted phenyl, p-methylphenyl, m-methylphenyl,p-methoxyphenyl, m-chlorophenyl, p-chlorophenyl or m-nitrophenyl,disclosed in German Patent 2,444,322 and JP-A-50-58092.

(3) An antibacterial compound wherein R is an optionally substitutedimidazolyl disclosed in German Patents 2,261,693, 2,254,873 and2,215,999; an antibacterial compound wherein R is 5-nitro-2-furyl or5-nitro-2-thienyl disclosed in German Patent publications 2,161,586,2,161,587 and 2,113,438.

(4) The structure of a compound wherein Ra is H, Rb is cyclopropyl, E isa single bond and R is a p-(trifluoromethyl)phenyl disclosed in CASRegistry File under the codes RN=289651-68-9.

(5) an antihypertensive compound wherein Ra is a methyl, Rb is a2-hydroxy-propyl, E is a single bond and R is a 3-pyridyl disclosed inFarmaco. Ed. Sci., 34(4), 299-310 (1979).

In order to reduce a pain such as a lumbar pain or to reduce the risk ofbone fracture in a metabolic bone disease such as osteoporosis, it isrequired to increase the bone mass and the bone strength, and thus it ishighly desired to develop a clinically useful bone-forming agent havingan ability of stimulating the bone formation by osteoblasts which isconsidered to be surely effective.

SUMMARY OF THE INVENTION

As a result of an effort made by us for the purpose of developing atherapeutic agent having a bone formation-stimulating effect bypromoting the functions of osteoblasts, the present inventors discoveredthat a nitrogen-containing heterocyclic compound shown below exhibits apotent bone formation-stimulating effect on the osteoblast and thus canserve as an excellent prophylactic or therapeutic agent against ametabolic bone disease, whereby establishing the invention.

Thus, the invention relates to a nitrogen-containing heterocycliccompound represented by the formula (I):

or a pharmaceutically acceptable salt thereof, wherein

-   -   Ra and Rb: the same or different and each represent H; CO-lower        alkyl; SO₂-lower alkyl; an optionally substituted cycloalkyl; an        optionally substituted aryl; or a lower alkyl which may have 1        to 3 substituents selected from the group consisting of an        optionally substituted cycloalkyl, an optionally substituted        aryl, an optionally substituted 4- to 8-membered monocyclic        saturated or partially unsaturated heterocyclic ring, CO-lower        alkyl, SO₂-lower alkyl, OR¹, SR¹, NR¹R², halogen, NO₂, CN and        COOR¹; provided that at least one of Ra and Rb represent a group        other than H; or,    -   Ra and Rb taken together with an adjacent N atom represent a 4-        to 8-membered saturated or partially unsaturated heterocyclic        ring containing 1 to 2 nitrogen atoms as heteroatoms, said        heterocyclic ring may be fused with a benzene ring or a        cycloalkyl ring and may have A bridge and may form a spiro ring,        and said heterocyclic ring may have 1 to 5 substituent,    -   E: a single bond, C₁₋₃ alkylene, vinylene (—C═C—), ethynylene        (—C≡C—), CO, NR³, CH₂-J, CONR⁴ or NR⁵CO,    -   J: O, S, NR⁶, CO, SO or SO₂,    -   R: an optionally substituted aryl, an optionally substituted        heteroaryl, an optionally substituted cycloalkyl, an optionally        substituted cycloalkenyl or an optionally substituted 4- to        8-membered monocyclic saturated or partially saturated        heterocyclic ring,    -   R¹ to R⁶: the same or different and each denotes H or lower        alkyl;    -   with the proviso that the following compounds are excluded:    -   (1) a compound wherein Ra and Rb taken together with an adjacent        N atom represent a piperidino, E is a single bond and R is a        piperidino, unsubstituted phenyl, p-(trifluoromethyl)phenyl,        p-chlorophenyl or o-nitrophenyl,    -   (2) a compound wherein Ra and Rb taken together with an adjacent        N atom represent a 4-methyl-1-piperazinyl, E is a single bond,        and R is an unsubstituted phenyl, p-methylphenyl,        m-methylphenyl, p-methoxyphenyl, m-chlorophenyl, p-chlorophenyl        or m-nitrophenyl,    -   (3) a compound wherein R is an optionally substituted        imidazolyl, 5-nitro-2-furyl or 5-nitro-2-thienyl,    -   (4) a compound wherein Ra is H, Rb is cyclopropyl, E is a single        bond and R is a p-(trifluoromethyl)phenyl, and    -   (5) a compound wherein Ra is a methyl, Rb is a 2-hydroxypropyl,        E is a single bond and R is a 3-pyridyl, and the same applies        analogously to the followings.

The present invention also relates to a pharmaceutical compositioncomprising a nitrogen-containing heterocyclic compound represented bythe formula (I) shown above or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier, especially to abone-forming agent. Furthermore, the invention encompasses use of anitrogen-containing heterocyclic compound represented by the formula (I)or a pharmaceutically acceptable salt thereof for manufacturing abone-forming agent for a mammalian animal as well as a method forstimulating bone formation in a mammalian animal comprisingadministering an effective amount of a nitrogen-containing heterocycliccompound represented by the formula (I) or a pharmaceutically acceptablesalt thereof to the mammalian animal.

A compound represented by the formula (I) is detailed below.

As used herein, a “lower” means, unless otherwise specified, a straightor branched carbon chain having 1 to 6 carbon atoms. A “lower alkyl” ispreferably methyl, ethyl and propyl group. In this specification, “Alk”is an abbreviation of “lower alkyl”.

An “aryl” is preferably a C₆₋₁₄ monocyclic to tricyclic aryl group. Morepreferably, it is a phenyl or naphthyl group, particularly, a phenylgroup. It is also possible that a phenyl group is fused with a C₅₋₈cycloalkyl group to form, for example, an indanyl or tetrahydronaphthylgroup. A “cycloalkyl” is preferably a C₃₋₁₄ cycloalkyl group, which mayhave bridge(s). More preferably, it is a C₃₋₁₀ cycloalkyl group,particularly, a cyclopentyl, cyclohexyl and cycloheptyl group. A“cycloalkenyl” is a group having 1 to 2 double bonds in theabove-mentioned “cycloalkyl” ring.

A “4- to 8-membered monocyclic saturated or partially unsaturatedheterocyclic ring” is a 4- to 8-membered monocyclic saturatedheterocyclic ring having 1 to 4 heteroatoms selected from N, S and O,which may have bridge(s) and may partially have an unsaturated bond.Preferably, it is tetrahydropyranyl, tetrahydrofuranyl, pyrrolidinyl,piperidinyl, 1,2,3,6-tetrahydropyridyl, homopiperidinyl, piperazinyl,homopiperazinyl, quinucridinyl and morpholinyl group.

A “heteroaryl” is a 5- to 6-membered monocyclic heteroaryl group having1 to 4 heteroatoms selected from N, S and O, which may be fused with abenzene ring or a 5- to 6-membered monocyclic heteroaryl to form a bi-to tri-cyclic heteroaryl group, which may be saturated partially. Such a5- to 6-membered heteroaryl is preferably a furyl, thienyl, pyrrolyl,imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl,pyridazinyl, pyrazinyl and triazinyl group, while a bi- to tri-cyclicheteroaryl is preferably a benzofuranyl, benzothienyl,benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl,benzoimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl,cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, pyrazinopyridyl,triazolopyridyl, naphthylidinyl and imidazopyridyl group. A partiallysaturated heteroaryl may for example be 1,2,3,4-tetrahydroquinolylgroup. More preferably, it is a pyridyl, pyrimidinyl, furyl, thienyl,thiazolyl, quinolyl, benzofuranyl, benzothienyl, indolyl, imidazopyridyland naphthylidinyl group, especially a pyridyl group.

A substituent on an “optionally substituted aryl”, and “optionallysubstituted heteroaryl”, “optionally substituted cycloalkyl”,“optionally substituted cycloalkenyl”, “optionally substituted 4- to8-membered monocyclic saturated or partially unsaturated heterocyclicring” is preferably the same or different 1 to 5 substituents selectedfrom Group B shown below, more preferably groups selected from Group B1,especially a halogen, OAlk and SAlk.

Group B: An Alk which may have 1 to 4 substituents selected from GroupG, halogen, NR¹R², NR¹CO-Alk, NO₂, CN, OR¹, —O-(Alk having 1 to 4substituents selected from Group G), SR¹, —S-halogeno-Alk, —O—CO-Alk,COOR¹, COR¹, CONR¹R², SOAlk, SO₂Alk, SO₂NR¹R², P(═O) (OR¹)₂, —O—CH₂—O—,—O—(CH₂)₂—O—, aryl which may have 1 to 4 substituents selected fromGroup D, heteroaryl which may have 1 to 4 substituents selected fromGroup D, —O-(aryl which may have 1 to 4 substituents selected from GroupD), 4- to 8-membered monocyclic saturated or partially unsaturatedheterocyclic ring which may have 1 to 4 substituents selected from GroupD, cycloalkyl and —O-cycloalkyl. In these groups, R¹ and R² are asdefined above; “Group D” consists of Alk, halogen, halogeno-Alk, NR¹R²,NO₂, CN, OR¹ and SR¹; “Group G” consists of halogen, NR¹R², CN, COOR¹,OR¹, SR¹, 4- to 8-membered monocyclic saturated or partially unsaturatedheterocyclic ring which may have 1 to 4 substituents selected from GroupD, aryl which may have 1 to 4 substituents selected from Group D andheteroaryl which may have 1 to 4 substituents selected from Group D; a“halogen” is I, Br, F and Cl; and a “halogeno-Alk” is a lower alkylsubstituted by 1 or more halogen atoms (especially CF₃). The sameapplies analogously to the followings.

Group B1: Alk, halogen, halogeno-Alk, NR R², NO₂, CN, OR¹,—O-halogeno-Alk, SR¹, COOR¹, CONR¹R², SO₂Alk, 4- to 8-memberedmonocyclic saturated or partially unsaturated heterocyclic ring, phenyland phenoxy group.

A “4- to 8-membered saturated or partially unsaturated heterocyclicring” which may be formed from Ra and Rb taken together with an adjacentN atom may for example be a 4- to 8-membered monocyclic saturated orpartially unsaturated heterocyclic ring having 1 to 2 N atoms as ringatoms with the rest of the ring atoms being C atoms. Such a heterocyclicring may form a fused ring together with a benzene ring or a C₅₋₈cycloalkyl ring, may have bridge(s), and may form a Spiro ring.Preferably, it is pyrrolydinyl, piperidinyl, homopiperidinyl,piperazinyl, pyrazolidinyl, imidazolidinyl, homopiperazinyl,perhydroazocinyl, pyrrolinyl, imidazolinyl, pyrazolinyl,1,2,3,6-tetrahydropyridyl, 1,2-dihydropyridyl, tetrahydropyridazinyl,tetrahydropyrazinyl, 1,4,5,6-tetrahydropyrimidinyl, indolinyl,isoindolinyl, 1,2,3,4-tetrahydroquinolyl, 1,2,3,4-tetrahydroisoquinolyl,3-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl,3-azabicyclo[3.2.2]nonyl, 3-azabicyclo[3.3.1]nonyl,7-azabicyclo[2.2.1]heptyl, isoquinucridinyl, 3-azabicyclo[3.3.2]decanyl,3-azaspiro[5.5]undecanyl, 2-azaspiro[4.5]decanyl, 2-azaspiro[4.4]nonyland 8-azaspiro[4.5]decanyl and the like. It is more preferably a 4- to8-membered monocyclic saturated or partially unsaturated heterocyclicring which may have bridge(s), especially, pyrrolidinyl, piperidinyl,homopiperidinyl, perhydroazocinyl, 1,2,3,6-tetrahydropyridyl,3-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl,3-azabicyclo[3.2.2]nonyl and 3-azabicyclo[3.3.1]nonyl group. A piperidylgroup is especially preferred.

Such a heterocyclic ring may have substituent(s), and such a substituentis preferably 1 to 5 substituents selected from Group B listed above.More preferably, it is 1 to 5 substituents selected from (Alk which mayhave substituent(s) selected from COOR¹, OR¹ and phenyl), halogen,NR¹R², CN, OR¹, —O-(Alk which may have substituent(s) selected fromCOOR¹, OR and phenyl), SR COOR¹, CONR¹R² and phenyl, especially 1 to 2substituents selected from Alk, halogen, OR¹ and COOR¹.

Among the invention compounds (I), those preferred are listed below.

-   -   (1) A compound wherein —NRaRb forms a 4- to 8-membered saturated        or partially unsaturated heterocyclic ring having 1 to 2 N atoms        as heteroatoms which may be fused with a benzene ring or a        cycloalkyl ring, which may have bridge(s), or which may form        spiro ring(s) and which may have 1 to 5 substituents selected        from Group B; E is a single bond, C₁₋₃ alkylene, vinylene,        ethynylene, CONH, CH₂NH, CH₂O or CH₂S; R is aryl which may have        1 to 5 substituents selected from Group B or heteroaryl which        may have 1 to 5 substituents selected from Group B.    -   (2) A compound wherein E is a single bond, C₁₋₃ alkylene,        vinylene or ethynylene; R is an aryl having 1 to 5 substituents        selected from Group B1 or heteroaryl having 1 to 5 substituents        selected from Group B1.    -   (3) A compound wherein —NRaRb is a 4- to 8-membered monocyclic        saturated or partially unsaturated heterocyclic ring which may        have one N atom as a ring heteroatom and may have a bridge, and        may have 1 to 2 substituents selected from Alk, halogen, OR¹ and        COOR¹; E is a single bond; R is a phenyl having, at its        m-position, a substituent selected from a halogen, OAlk and SAlk        or a pyridyl having, at its 6-position, a substituent selected        from a halogen, OAlk and SAlk.

An especially preferred compound in the invention is nitrogen-containingheterocyclic compounds listed below and their pharmaceuticallyacceptable salts.

6-Azocan-1-yl-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azepan-1-yl-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,3-(3-methoxyphenyl)-6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine,3-(3-bromophenyl)-6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azepan-1-yl-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(4-fluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(3-azabicyclo[3.2.1]octan-3-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(4,4-difluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(3,3-difluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azocan-1-yl-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,and6-(8-azabicyclo[3.2.1]octan-8-yl)-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine.

Some of the invention substituents may allow geometric isomers ortautomers to exist, and the invention encompasses all these isomers asbeing separated or in a mixture. An invention compound may have anasymmetric carbon atom, based on which an optical isomer may exist. Theinvention encompasses all of these optical isomers as mixtures orindividually separated forms.

An invention compound (I) may form an acid addition salt or a salt witha base depending on the type of the substituent. Such a salt is apharmaceutically acceptable salt, preferably an acid addition salt withan inorganic acid such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and thelike and with an organic acid such as formic acid, acetic acid,propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,maleic acid, lactic acid, malic acid, tartaric acid, citric acid,methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acidand the like, a salt with an inorganic base including a metal such assodium, potassium, magnesium, calcium, aluminum and the like, and withan organic base such as methylamine, ethylamine, ethanolamine, lysine,ornithine and the like, as well as an ammonium salt.

Furthermore, the invention includes various hydrates or solvates of aninvention compound (I) or its salt, as well as the forms of polymorphiccrystals.

(Production Methods)

A representative method for producing an invention compound (I) isdescribed below.

An invention compound and a pharmaceutically acceptable salt thereof canbe produced by utilizing the characteristics based on its skeleton andthe types of the substituents and applying various known syntheticmethods. In such a case, it is advantageous sometimes from amanufacturing technological point of view that a certain functionalgroup in a starting material or an intermediate is substituted by asuitable protective group, i.e., a group which can readily be convertedback to this certain functional group. Thereafter, the protective groupis removed if necessary to obtain an intended compound. Such afunctional group may for example be a hydroxyl group or carboxyl group,and its protective group may be those listed for example in Greene andWuts, Protective Groups in Organic Synthesis, 2nd Ed., and can be usedappropriately depending on the reaction conditions.

A representative method for producing an invention compound is describedbelow.1st Preparation Method

wherein L is a leaving group such as a halogen atom or an organicsulfonate. the same applies analogously to the followings.

An invention compound (I) can be obtained by a standard N-alkylationmethod, for example by reacting an amine derivative (III) and a compound(II) having an ordinary leaving group such as a halogen atom or anorganic sulfonate in the presence or absence of a base such as potassiumcarbonate, triethylamine, sodium hydride and the like, in an inertsolvent such as N,N-dimethylformamide (DMF), toluene, tetrahydrofuran(THF), acetonitrile and the like or without using any solvent withcooling or under reflux.

Other Preparation Method

A compound obtained in the first preparation method described above canfurther be subjected to a standard substituent-modifying reaction, forexample, reduction from a nitro group to an amino group, amidation,sulfonamidation, N-alkylation, esterification, ester hydrolysis,hydroxyl group etherification, thioether sulfonation, halogenation,olefin-derivatization, and the like to obtain an invention compoundhaving a desired substituent. Any of these reactions can readily beconducted in accordance with a method described for example in ORGANICFUNCTIONAL GROUP PREPARAQTIONS Second Edition (Sandler, Karo).

(Method for Preparing Starting Material)

Preparation Method A

The starting material (II) of the present application can be prepared bysubjecting a hydrazine compound (IV) and a carboxylic acid compound (V)to a dehydration condensation reaction to form a hydrazide compound (VI)followed by a cyclization.

The dehydration condensation reaction in the first step can be conductedby a standard method, for example by using a free carboxylic acid and acoupling agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(WSCD) or a carboxylic acid-activating agent such as1,1′-carbonyldiimidazole, or using a reactive derivative of a carboxylicacid (for example, acid halide such as acid chloride and acid bromide;acid azides; active esters prepared from methanol, ethanol, benzylalcohol, optionally substituted phenol, N-hydroxysuccinimide and thelike; symmetric acid anhydrides; mixed acid anhydrides withalkylcarbonates, p-toluenesulfonic acid and the like).

The reaction is conducted using equimolar amounts or an excessive amountof any one of the reactants, in an organic solvent which is inert to thereaction, such as pyridine, THF, methylene chloride, DMF, acetonitrileand the like. The reaction temperature is selected appropriatelydepending on the type of the reactive derivative. In a case of a certainreactive derivative, it may be advantageous to add a base such as4-dimetylaminopyridine for promoting the reaction.

The cyclization in the second step can be conducted by a reaction in thepresence or absence of an acid such as acetic acid, p-toluenesulfonicacid, hydrochloric acid and the like, in a solvent such as xylene,ethylene glycol and the like, or without using any solvent. Thisreaction can be conducted at room temperature or with heating underreflux.Preparation Method B

An starting material (IX) of the present application can be produced bycoupling a compound (IV) with an isocyanate compound (VII) in a solventinert to the reaction such as acetonitrile to form a compound (VIII)followed by adding 1,2-dibromo-1,1,2,2-tetrachloroethane andtriphenylphosphine in the presence of a base such as triethylaminewhereby effecting a cyclization. The reaction can be conductedappropriately by a standard method at room temperature or with heatingunder reflux.

A reaction product obtained by each preparation method described abovecan be isolated and purified as a free base, free acid, its salt,hydrate or any of various solvates. A salt can be produced by anordinary salt formation reaction. The isolation and the purification canbe conducted by applying ordinary chemical procedures such asextraction, concentration, distillation, crystallization, filtration,recrystallization, various chromatographies and the like. Each isomercan be isolated utilizing a physicochemical difference between isomers.For example, an optical isomer can be separated by an ordinary opticalresolution method, for example, fractional crystallization orchromatography. An optical isomer can be synthesized from a suitableoptically active starting compound.

INDUSTRIAL APPLICABILITY

An invention compound has an ability of stimulating the bone formationby osteoblasts, and is useful in preventing or treating a metabolic bonedisease associated with a lower bone formation ability relative to thebone resorption ability. Such a metabolic bone disease includesosteoporosis, osteitis fibrosa (hyperparathyroidism), osteomalacia andPaget's disease which affects the parameters of systemic bonemetabolism. It is useful especially in a senile osteoporosis associatedwith a reduced bone formation ability.

An invention bone-forming agent may also be useful in promoting a curefrom a bone disease in the field of orthopedics such as bone fracture,bone loss and osteoarthritis, as well as in the field of dentistry fortreating a periodontitis or stabilizing an artificial dental root.

A phenotypic trait of osteoblasts includes an alkaline phosphatase (ALP)activity, production of bone matrix proteins (collagen, osteocalcin,osteonectin, osteopontin and the like), presence of an active vitamin D3receptor, parathyroid hormone receptor, estrogen receptor, androgenreceptor (Molecular Medicine, Vol. 30, No. 10, 1232 (1993)). The ALP isincreased at an early stage of the onset of the osteoblast functions(Journal of Cellular Physiology, Vol. 143, 420 (1990)). The role of theALP for the bone formation by the osteoblast is believed to be toincrease the phosphate ion level at a site of the bone formation and todecompose pyrophosphoric acid which is an inhibitor of calcification(“SAIBO KOGAKU”, Vol.13, No. 12, 1062 (1994)). It was also reported thata subcutaneous implantation of the ALP bound covalently to a collagensheet caused the calcification (J. Clin. Invest., 89, 1974 (1992)).Accordingly, the osteoblast-induced ALP activity elevation can beregarded as an index of the bone formation.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the osteoid thickness in an invention compound treatmentgroup and a control group in Experimental Example 2.

FIG. 2 shows the bone formation rate in an invention compound treatmentgroup and a control group in Experimental Example 2.

BEST MODE FOR CARRYING OUT THE INVENTION

The tests for exhibiting the pharmacological effects of inventioncompounds are described below together with their results.

EXPERIMENTAL EXAMPLE 1 Alkaline Phosphatase (ALP) Activity Measurementin Mouse Osteoblast Cell Line

A mouse osteoblast cell line MC3T3-E1 was seeded at the density of 3000cells/well in 96-well plates in 5% fetal bovine serum (FBS)-supplementedα-minimum essential medium (MEM) and incubated for 4 to 6 hours. To thecell thus cultured, a test compound dissolved in dimethyl sulfoxide(DMSO) (final concentration of DMSO: 0.5%) was added, and the incubationwas continued for further 3 days. After washing the cells with aphosphate-buffered physiological saline, the substrate was added andincubated at 37° C. for 10 to 15 minutes. The reaction was stopped byadding 0.5M sodium hydroxide, and the absorbance at a wavelength of 405nm (correction wavelength: 492 nm) was measured and represented by a %value based on the value in the control group being regarded as 100%,from which the ALP activity was calculated. The measurement describedabove was in accordance with the method by Lowry et al (Journal ofBiological Chemistry, Vol. 207, page 19, (1954)).

The invention compounds of Examples 2, 8, 12, 21, 23, 24, 27, 28, 33,34, 35, 38, 39, 42, 45, 54, 65, 67, 72, 75, 76, 81, 84, 85, 86, 87, 92,93, 94, 95, 96, 100, 103, 107, 114, 116, 117 and 118 exhibited the ALPactivities of 300% or higher at 300 nM when compared with the controlgroup.

EXPERIMENTAL EXAMPLE 2 Bone Formation Stimulation Test of Rat TopicalAdministration

The compound of Example 23 according to the present invention (1, 10 or100 μg) was dispersed in 0.1 ml of a mineral oil and given successivelyfor 10 days once a day using a 1 ml syringe fitted with a 25G injectionneedle subcutaneously to the right temporal region of each of 10-weekold male Wistar rats (n=6) under anesthesia with ether. In a controlgroup, each rat was treated only with 0.1 ml of the mineral oil once aday for successive 10 days. On the 11th day of the treatment, the drugwas discontinued and on the 18th day each rat was sacrificed to removethe calvaria. Before sacrificing the animal, tetracyclin (25 mg/kg) wasgiven on the 10th day of the treatment and then calcein (20 mg/kg) wasgiven subcutaneously to the dorsal area on the 16th day for labeling thebone. The calvaria thus obtained was fixed in 70% ethanol, and coronalsections were made according to a standard procedure and subjected to abone morphometric measurements. Osteoids thickness and bone formationrate in an invention compound treatment group and a control group areshown in FIG. 1 and FIG. 2.

As a result of the administration of the invention compounds,significant increases in osteoid thickness and bone formation rate wereobserved, exhibiting excellent bone formation-stimulating effects of theinvention compounds.

A pharmaceutical composition containing an invention compound (I) orpharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier can be prepared by an ordinary method using one ormore of a compound represented by the formula (I) or pharmaceuticallyacceptable salt thereof together with a pharmaceutical carrier,excipient and other additives employed usually in a formulation. Theadministration can be conducted in various dosage forms orally as atablet, pill, capsule, granule, powder, liquid, inhalation formulationand the like, or parenterally as an injection formulation such as anintravenous injection, intramuscular injection and the like, as well asa suppositories, percutaneous liquid formulations, ointments,percutaneous patches and the like.

A solid composition for an invention oral administration may be atablet, powder, granule and the like. In such a solid composition, oneor more active substance is mixed with at least one inert diluent suchas lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystallinecellulose, starch, polyvinylpyrrolidone, magnesium aluminatemetasilicate and the like. The composition may contain additives otherthan the inert diluent as customary in the art, including a lubricantsuch as magnesium stearate, a disintegrant such as calciumfibrinoglycolate, a stabilizer, a dissolution aid such as glutamic acidor aspartic acid. A tablet or pill may be sugar-coated if necessary withsucrose, gelatin, hydroxypropyl cellulose, hydroxypropylmethyl cellulosephthalate and the like or covered with a gastric or enteric filmcoating.

A liquid composition for an oral administration contains apharmaceutically acceptable emulsifier, solubilizer, suspending agent,syrup, elixir and the like, together with an ordinarily employed inertdiluent such as a purified water and ethanol. Such a composition maycontain, in addition to an inert diluent, other auxiliary agents such asa humectant, suspending agent, as well as a sweetener, flavor, fragranceand preservative.

An injection formulation for a parenteral administration includes anaseptic aqueous or non-aqueous solution, suspension and emulsion. Suchan aqueous solution and suspension may contain a distilled water forinjection or a physiological saline. A non-aqueous solution andsuspension may contain propylene glycol, polyethylene glycol, avegetable oil such as an olive oil, an alcohol such as ethanol, as wellas Polysorbate 80 (trade name) and the like. Such a composition may alsocontain auxiliary agents such as a preservative, humectant, emulsifier,dispersing agent, dissolution aid (for example, glutamic acid, asparticacid) and the like. Any of these materials can be made aseptic byfiltration through a filter retaining bacteria, incorporation of asterilizing agent or irradiation. Any of these material can beformulated as an aseptic solid composition which is to be reconstitutedjust before use with an aseptic water or aseptic solvent for injection.

In a usual oral administration, the daily dose is about 0.001 to 10mg/kg body weight, preferably 0.01 to 5 mg/kg, which may be given atonce, or twice to four times in portions. When given intravenously, thedaily dose is about 0.0001 to 1 mg/kg body weight, which may be given atonce, or several times in portions. The dosage may vary depending on thesymptom, age, sex and the like of each individual patient.

EXAMPLES

The invention is further described in the following Examples, which arenot intended to restrict the invention compositions. The ReferenceExamples indicate methods for producing starting materials.Abbreviations in the text are Dat: physicochemical characteristics, F:FAB-MS (M+H)⁺; other symbols are as defined above.

Reference Example 1

A mixture of (6-chloropyridazin-3-yl)hydrazine, 4-nitrobenzoic acid,WSCD hydrochloride and THF was stirred at room temperature for 2 hours.The reaction solution was combined with water, and the precipitate wascollected by filtration, washed with water and diethyl ether to obtainN′-(6-chloropyridazin-3-yl)-4-nitrobenzohydrazide. To this, acetic acidwas added, and the mixture was stirred at 110° C. for 2 hours, thereaction solution was concentrated under reduced pressure, and theresultant crude crystal was washed with ethanol to obtain6-chloro-3-(4-nitrophenyl)-1,2,4-triazolo[4,3-b]pyridazine. Dat (F:276).

Reference Example 2

To a solution of (6-chloropyridazin-3-yl)hydrazine and triethylamine inTHF, 3-cyanobenzoyl chloride was added with cooling in ice, and stirredat room temperature for 1 hour. The reaction solution was combined withwater, and the precipitate was collected by filtration, washed withwater and diethyl ether to obtainN′-(6-chloropyridazin-3-yl)-3-cytanobenzohydrazide. To this, acetic acidwas added, and the mixture was stirred at 110° C. for 2 hours, thereaction solution was concentrated under reduced pressure, and theresultant crude crystal was washed with ethanol to obtain3-(6-chloro-1,2,4-triazolo[4,3-b]pyridazin-3-yl)benzonitrile. Dat(F:256).

Reference Example 3

A mixture of (6-chloropyridazin-3-yl)hydrazine, 3-dimethylaminobenzoicacid, WSCD hydrochloride and THF was stirred at room temperature for 2hours. The reaction solution was combined with water, and theprecipitate was collected by filtration, washed with water and isopropylether to obtainN′-(6-chloropyridazin-3-yl)-3-dimethylaminobenzohydrazide. To this,ethylene glycol was added and the mixture was stirred at 160° C. for 4hours. After allowing to cool to room temperature, followed bypurification by a standard method,6-chloro-3-(3-dimethylaminophenyl)-1,2,4-triazolo[4,3-b]pyridazine wasobtained. Dat (F:274).

Reference Example 4

A solution of 3-methoxyphenyl isocyanate and3-chloro-6-hydrazinopyridazine in acetonitrile was stirred at roomtemperature for 30 minutes, and 1,2-dibromo-1,1,2,2-tetrachloroethanewas added and the mixture was stirred at room temperature for further 2hours. This reaction solution was combined with triethylamine andtriphenylphosphine with cooling in ice, and stirred at room temperaturefor 3 days. Then, the reaction solution was concentrated under reducedpressure, purified by a standard method to obtain6-chloro-N-(3-methoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazin-3-amine. Dat(F:276).

Similarly to Reference Example 1, the compounds of Reference Examples 5to 50 indicated in Tables 1 to 2 shown below were obtained.

Example 1

A mixture of3-(6-chloro-1,2,4-triazolo[4,3-b]pyridazin-3-yl)benzonitrile (450 mg)and piperidine (5 ml) was stirred with heating under reflux for 2 hours.The reaction solution was concentrated under reduced pressure, and theresultant residue was extracted with chloroform. The extract was washedwith saturated aqueous solution of ammonium chloride and brine, driedover anhydrous magnesium sulfate, and concentrated under reducedpressure. The resultant crude crystals were recrystallized from ethanolto obtain3-[6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl)benzonitrile(435 mg) as slightly yellowish crystals.

Example 2

To a solution ofN-cyclopentyl-3-(3-methoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazine-6-amine(300 mg) in DMF (5 ml), 60% sodium hydride (44 mg) was added withcooling in ice, and the mixture was stirred at an ice-coolingtemperature to room temperature for 1 hour, combined with methyl iodide(68 μl), and stirred further for 2 hours. The reaction mixture wascombined with water, and extracted with ethyl acetate. The extract waswashed with brine and dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The resultant residue was purifiedby silica gel column chromatography (eluent: chloroform:methanol=20:1),recrystallized from ethyl acetate to obtainN-cyclopentyl-3-(3-methoxyphenyl)-N-methyl-1,2,4-triazoro[4,3-b]pyridazine-6-amine(145 mg) as pale yellow crystals.

Example 3

To a mixture of potassium hydroxide (98 mg) and DMSO (5 ml),6-azepan-1-yl-3-(1H-indol-2-yl)-1,2,4-triazolo[4,3-b]pyridazine (501 mg)was added, and the mixture was stirred at room temperature for 30minutes, and then combined with methyl iodide (0.15 ml) and then stirredfor further 2 hours at room temperature. The reaction mixture wascombined with water, and the resultant solids were collected byfiltration, washed with a solvent mixture of water and methanol, andthen purified by silica gel column chromatography (eluent:chloroform:methanol=99:1). The resultant crude crystals wererecrystallized from ethanol to obtain6-azepan-1-yl-3-(1-methylindol-2-yl)-1,2,4-triazolo[4,3-b]pyridazine (40mg) as colorless crystals.

Example 4

A mixture of3-[6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine-3-yl]aniline (180mg) and acetic anhydride (3 ml) was stirred at room temperature for 6hours. The reaction solution was concentrated under reduced pressure,and the resultant crude crystals were washed with diethyl ether toobtain3′-[6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]acetoanilide(190 mg) as colorless crystals.

Example 5

To a solution mixture of6-piperidin-1-yl-3-piperidin-3-yl-1,2,4-triazolo[4,3-b]pyridazine (670mg), triethylamine (360 mg) and methylene chloride (15 ml),methanesulfonyl chloride (320 mg) was added and the mixture was stirredat room temperature for 8 hours. The reaction solution was concentratedunder reduced pressure, and the resultant residue was extracted withethyl acetate. The extract was washed successively with water and brine,dried over anhydrous magnesium sulfate, and concentrated under reducedpressure. The resultant crude crystals were recrystallized from ethanolto obtain3-(1-methanesulfonylpiperidin-3-yl)-6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazine(230 mg) as colorless crystals.

Example 8

A mixture of6-azocan-1-yl-3-(6-chloropyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine(360 mg), sodium methoxide (570 mg) and toluene (20 ml) was stirred withheating under reflux for 3 hours. After allowing to cool to roomtemperature, the reaction solution was concentrated under reducedpressure, and the resultant residue was extracted with ethyl acetate.The extract was washed with water and brine, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. Theresultant crude crystal was washed with diethyl ether to obtain6-azocan-1-yl-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine(290 mg) as pale yellow crystals.

Example 9

To a solution mixture of3-[6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine-3-yl]benzoic acid(430 mg), a catalytic amount of DMF and THF (10 ml), oxalyl chloride(0.44 ml) was added with cooling in ice, and the mixture was stirred atroom temperature for 2 hours and then ammonia gas was passed withcooling in ice for 15 minutes. The reaction solution was concentratedunder reduced pressure, and the resultant residue was combined withchloroform:methanol (10:1), and then insoluble materials were filteredoff. The filtrate was concentrated under reduced pressure, and theresultant crude crystals were recrystallized from ethanol to obtain3-[6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]benzamide (242mg) as slightly tan crystals.

Example 10

To a solution of3-[3-(methylsulfanyl)phenyl]-6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazine(550 mg) in methylene chloride (30 ml), 3-chloroperbenzoic acid (1.25 g)was added at room temperature, and the mixture was stirred for 13 hours.The reaction solution was combined with water, and diluted withmethylene chloride. The organic phase washed successively with water, 1Maqueous solution of sodium hydroxide and brine, dried over anhydroussodium sulfate, and concentrated under reduced pressure. The resultantresidue was purified by silica gel column chromatography (eluent:chloroform:methanol=98:2) and washed with ethanol to obtain3-[3-(methylsulfonyl)phenyl]-6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazine(250 mg) as colorless crystals.

Example 11

A solution mixture of concentrated sulfuric acid (1.5 ml) and water (3ml) was cooled to −5° C., and4-(6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)-1,3-thiazol-2-amine(0.90 g), copper (II) sulfate (1.50 g) and sodium bromide (0.62 g) wereadded successively, and the mixture was stirred at 0° C. for 5 minutes.Then, a solution of sodium nitrite (0.25 g) in water (1.6 ml), followedby stirring at room temperature overnight. The reaction solution wascombined with water, chloroform and 2-propanol, and insoluble materialswere filtered off. The resultant organic phase was washed with water,dried over anhydrous magnesium sulfate, and concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (eluent: chloroform:methanol=20:1) to obtain crudecrystals, which were then recrystallized from ethanol-diethyl ether toobtain3-(2-bromo-1,3-thiazol-4-yl)-6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazine(75 mg) as pale yellow crystals.

Example 12

A mixture of6-(6-azepan-1-yl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)pyridine-2-ol (700mg) and phosphorus tribromide (7 ml) was stirred at 130° C. for 6 hours.After allowing to cool to room temperature, an ice-water was added, andthe mixture was neutralized with saturated aqueous solution of potassiumcarbonate, and extracted with chloroform. The extract was washed withsaturated aqueous solution of sodium hydrogen carbonate and brine, driedover anhydrous sodium sulfate, and then concentrated under reducedpressure. The resultant residue was purified by silica gel columnchromatography (eluent: chloroform:methanol=98:2) and recrystallizedfrom ethanol to obtain6-azepan-1-yl-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine(240 mg) as colorless crystals.

Example 13

To a solution of diethyl[(6-azepan-1-yl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)methyl]phosphonate(367 mg) in THF (10 ml), potassium tert-butoxide (127 mg) was added withcooling in an ice bath, and the mixture was stirred at room temperaturefor 40 minutes. The resultant red solution was combined with2-bromobenzaldehyde (0.128 ml), and stirred at room temperature forfurther 1 hour. The reaction solution was combined with water, andextracted with ethyl acetate. The extract was washed with water andbrine, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure. The resultant white solids were recrystallized frommethanol to obtain6-azepan-1-yl-3-[(E)-2-(2-bromophenyl)vinyl]-1,2,4-triazolo[4,3-b]pyridazine(152 mg) as colorless crystals.

Example 14

A mixture of ethyl6-chloro-1,2,4-triazolo[4,3-b]pyridazine-3-carboxylate (3.00 g) andhexamethylenimine (10 ml) was stirred at 100° C. for 2 hours. Thereaction solution was concentrated under reduced pressure, and theresultant residue was purified by silica gel column chromatography(eluent: chloroform:methanol=30:1) to obtain crude crystals which werethen recrystallized from ethanol and diethyl ether to obtain3-(azepan-1-ylcarbonyl)-6-azepan-1-yl-1,2,4-triazolo[4,3-b]pyridazine(0.23 g) as colorless crystals.

Example 15

A mixture of 1-[(benzyloxy)carbonyl]piperidine-3-carboxylic acid (2.44g), 3-chloro-6-hydrazinopyridazine (1.34 g), WSCD hydrochloride (2.13 g)and methylene chloride (60 ml) was stirred at room temperature for 16hours. The reaction solution was concentrated under reduced pressure andextracted with ethyl acetate. The extract was washed successively withwater and brine, dried over anhydrous sodium sulfate, and concentratedunder reduced pressure. The resultant reside was combined with aceticacid (80 ml), stirred at 100° C. for 2 days, and then the solvent wasdistilled off under reduced pressure. The resultant crude crystals werewashed with ethanol, and stirred with heating under reflux in piperidine(10 ml) for 3 hours. The reaction solution was concentrated underreduced pressure, and purified by silica gel column chromatography(eluent: chloroform:methanol=97:3). The resultant colorless solids werecombined with ethanol (40 ml) and 10% palladium-carbon (150 mg), andstirred under hydrogen atmosphere at room temperature for 6 hours, andthen the catalyst was filtered off. The resultant filtrate wasconcentrated under reduced pressure to obtain6-piperidin-1-yl-3-piperidin-3-yl-1,2,4-triazolo[4,3-b]pyridazine (1.18g) as a colorless amorphous.

Example 16

To a solution of ethyl 2-aminothiazole-4-carboxylate (4.56 g) in THF(200 ml), 1M aqueous solution of sodium hydroxide (30 ml) was added andthe mixture was stirred at room temperature for 3 hours. The reactionsolution was combined with 1M hydrochloric acid (30 ml), concentrated,and the resultant residue was dissolved in DMF (50 ml). Then,6-chloropyridazin-3-ylhydrazine (3.83 g) and WSCD hydrochloride (6.09 g)were added, and the mixture was stirred at room temperature. Thereaction solution was combined with water, and the precipitate wascollected by filtration, and washed with water and diethyl ether,combined with acetic acid (30 ml), heated under reflux, and then thereaction solution was concentrated under reduced pressure. The residuewas combined with saturated aqueous solution of sodium hydrogencarbonate, and extracted with ethyl acetate. The organic layer waswashed with water and brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. To this, piperidine (10 ml) wasadded and the mixture was heated at 100° C., and the reaction solutionwas concentrated under reduced pressure, and the resultant residue waspurified by silica gel column chromatography (eluent:chloroform:methanol=30:1) to obtain3-(2-aminothiazol-4-yl)-6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine(0.92 g) as yellow solids.

Example 17

To a solution of 6-hydrazino-N-methyl-N-phenylpyridazine-3-amine (1.14g) in methylene chloride (10 ml), 6-chloropicolic acid (0.83 g) and WSCDhydrochloride (1.22 g) were added, and the mixture was stirred at roomtemperature overnight. The reaction solution was purified by silica gelcolumn chromatography (eluent: chloroform) to obtain6-chloro-N′-{6-[methyl(phenyl)amino]pyridazin-3-yl}pyridine-2-carbohydrazide(0.57 g). This compound (0.56 g) was stirred at 150° C. overnight inxylene (20 ml), and the reaction solution was concentrated to obtain3-(chloropyridin-2-yl)-N-methyl-N-phenyl-1,2,4-triazolo[4,3-b]pyridazine-6-amine(0.54 g) as colorless solids.

Example 18

A mixture of6-chloro-3-(6-chloropyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine (620mg), heptamethylenimine (1.32 g) and 1,4-dioxane (20 ml) was stirred at100° C. for 9 hours. After allowing to cool to room temperature, thereaction solution was concentrated under reduced pressure, and theresultant residue was extracted with ethyl acetate. The extract waswashed successively with 5% aqueous solution of citric acid, water,saturated aqueous solution of sodium hydrogen carbonate and brine, driedover anhydrous sodium sulfate, and concentrated under reduced pressure.The resultant crude crystal was recrystallized from ethanol, to obtain6-azocan-1-yl-3-(6-chloropyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine(370 mg) as grayish white crystals.

Example 19

A mixture of6-(6-chloro-1,2,4-triazolo[4,3-b]pyridazin-3-yl)pyridin-2-ol (960 mg)and piperidine (10 ml) was stirred at 100° C. for 3 hours. Afterallowing to cool to room temperature, the reaction solution wasconcentrated under reduced pressure, and the resultant crystallineresidue was recrystallized from ethanol to obtain6-(6-piperidin-1-yl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)pyridin-2-ol(820 mg) as grayish white crystals.

By proceeding similarly to Example 1 optionally with conducting anordinary salt formation using 4M hydrogen chloride-ethyl acetate, thecompounds of Examples 20 to 170 were obtained. The compound of Example108 was obtained similarly to Example 2, the compound of Example 109similarly to Example 3, the compounds of Examples 110 and 111 similarlyto Example 4, the compounds of Examples 112 to 114 similarly to Example8, the compound of Example 115 similarly to Example 10, the compounds ofExamples 116 to 118 similarly to Example 12, the compounds of Examples119 and 120 similarly to Example 13, the compound of Example 121similarly to Example 18, and the compounds of Example 122 to 123similarly to Example 19.

The structures and the physicochemical characteristics of the compoundsof Reference Examples are indicated in Tables 1 to 2 shown below, andthe structures and the physicochemical characteristics of the compoundsof Examples are indicated in Tables 3 to 11. The compounds indicated inTable 12 can readily be produced using appropriate starting materialsalmost similarly to the methods described in Examples or PreparationMethod described above, with or without any modification obvious tothose skilled in the art.

The abbreviations in Tables are Rex: Reference Example number; Ex:Example number; Dat: physicochemical characteristics (F: FAB-MS (M+H)⁺;M: melting point [° C.]; (d): decomposition; Ni: NMR (DMSO-d₆, TMSinternal standard) characteristic peak δ ppm); Sal: salt (void: freebase; HCl: hydrochloride; 2HCl: dihydrochloride); Me: methyl; Et: ethyl;and Ac: acetyl. TABLE 1

Rex —E—R Dat 5

F: 261 6

N1: 3.93(3H, s), 7.60(1H, d, J=9.7 Hz), 8.93(1H, t, J=1.8 Hz) 7

F: 275 8

F: 323 9

F: 310 10

F: 381 11

F: 311 12

F: 357 13

F: 262 14

F: 227 15

N1: 7.37(1H, ddd, J=7.5, 4.7, 1.1 Hz), 7.59(1H, d, J=9.7 Hz), 8.00(1H,d, J=4.0 Hz) 16

F: 277 17

F: 315 18

F: 312 19

F: 259 20

F: 263 21

F: 248 22

F: 287 23

F: 308 24

N1: 2.64(3H, s), 7.28(1H, d, J=3.8 Hz), 7.57(1H, d, J=9.7 Hz) 25

F: 301 26

F: 238

TABLE 2 27

F: 270 28

F: 271 29

F: 267 30

F: 301 31

N1: 1.97-2.04(4H, m), 6.74(1H, dd, J=8.4, 2.4 Hz), 8.52(1H, d, J=9.7 Hz)32

E: 282 33

F: 298 34

F: 295 35

F: 337 36

F: 270 37

F: 8.17(1H, d, J=8.6 Hz), 8.62(1H, d, J=9.7 Hz), 8.94(1H, s) 38

N1: 7.09(1H, d, J=9.3 Hz), 8.60(1H, d, J=9.7 Hz), 8.69(1H, s) 39

F: 305 40

N1: 2.82(3H, s), 7.58(1H, d, J=9.7 Hz), 9.15(1H, s) 41

F: 289 42

F: 312 43

F: 304 44

F: 307 45

F: 279 46

F: 291 47

F: 327 48

F: 285 49

F: 279 50

F: 278

TABLE 3 (I)

Ex RbRaN— —E—R Sal Dat 1

M: 200-202 2

M: 136-137 N1: 2.52(3H, s), 3.86(3H, s), 8.03(1H, d, J=7.9 Hz) 3

M: 151-152 4

M: 210-212 5

M: 207-208 6

M: 153-154 7

2HCl N1: 4.00(3H, s), 4.82(2H, t, J=4.6 Hz), 8.42(1H, d, J=9.8 Hz) 8

M: 168-169 N1: 1.76(4H, br s), 3.98(3H, s), 6.94(1H, d, J=8.3 Hz) 9

M: 254-257(d) 10

M: 259-260 11

N1: 1.65(4H, br s), 8.15(1H, d, J=10.3 Hz), 8.67(1H, s) 12

M: 151-152 N1: 1.80(4H, br s), 7.34(1H, d, J=10.2 Hz), 7.75(1H, d, J=7.8Hz)

TABLE 4 13

M: 180-182 14

N1: 3.55-3.70(6H, m), 7.29(1H, d, J=9.8 Hz), 8.08(1H, d, J=10.2 Hz) 15

F: 421 16

F: 302 17

F: 337 18

F: 343 19

F: 297 20

M: 235-236 21

M: 165-166 N1: 3.85(3H, s), 7.07(1H, dd, J=7.9, 2.4 Hz), 7.43(1H, d,J=10.2 Hz) 22

M: 307-310 23

M: 207-208 N1: 1.68(6H, br), 7.50(1H, d, J=10 Hz), 9.49(1H, t, J=2.0 Hz)24

M: 168-169 25

M: 218-220 26

M: 167-169 27

M: 184-186 28

M: 189-191

TABLE 5 29

M: 165-167 30

M: 175-176 31

M: 135-136 32

M: 127-128 33

M: 172-173 N1: 3.85(3H, s), 7.27(1H, d, J=10.4 Hz), 7.48(1H, t, J=8.1Hz) 34

M: 152-153 N1: 1.66(6H, br s), 8.13(1H, d, J=10.3 Hz), 8.69(1H, s) 35

M: 138-139 N1: 1.79(4H, br s), 3.85(3H, s), 7.24(1H, d, J=10.2 Hz) 36

M: 181-182 37

M: 226-228 38

M: 158-159 N1: 1.66(6H, br s), 8.12(1H, d, J=10.2 Hz), 8.92(1H, s) 39

M: 163-164 N1: 3.97(3H, s), 6.94(1H, d, J=7.8 Hz), 7.45(1H, d, J=10.2Hz) 40

M: 123-124 41

M: 152-153 42

M: 185-186 N1: 2.56(3H, s), 7.43(1H, d, J=10.2 Hz), 8.38(1H, s) 43

M: 155-156

TABLE 6 44

M: 248-249 N1: 1.67(6H, br s), 7.54(1H, d, J=10.3 Hz), 8.65(1H, s) 45

M: 149-150 N1: 3.92(3H, s), 4.81(2H, s), 8.05(1H, d, J=7.8 Hz) 46

M: 104-105 47

M: 192-193 48

F: 339 N1: 0.91(6H, d, J=6.3 Hz), 3.99(3H, s), 8.13(1H, d, J=10.2 Hz) 49

N1: 3.97(3H, s), 6.95(2H, dd, J=7.8, 1.5 Hz), 8.14(1H, d, J=10.3 Hz) 50

M: 135-136 N1: 3.98(3H, s), 5.82(1H, d, J=10.2 Hz), 6.95(1H, d, J=7.9Hz) 51

N1: 3.97(3H, s), 4.44(1H, br s), 8.18(1H, d, J=10.2 Hz) 52

N1: 1.21(3H, t, J=6.8 Hz, 3.97(3H, s), 8.22(1H, d, J=10.3 Hz) 53

N1: 3.58(3H, s), 3.97(3H, s), 8.25(1H, d, J=10.3 Hz) 54

M: 118-119 N1: 1.51(4H, br s), 3.97(3H, s), 6.94(1H, d, J=8.3 HZ) 55

M: 196-197 N1: 1.79(4H, br s), 6.90(1H, d, J=3.4 Hz), 7.30(1H, d, J=10.2Hz) 56

M: 133-135 57

M: 244-246

TABLE 7 58

M: 226-228 59

M: 283-284 60

M: 307-309 61

M: 196-197 62

N1: 3.6-3.7(4H, m), 7.18(1H, d, J=10.3 Hz), 7.96(1H, d, J=10.3 Hz) 63

M: 154-155 N1: 3.87(3H, s), 8.05(1H, d, J=9.6 Hz), 8.52(1H, d, J=16.8Hz) 64

M: 191-192 65

F: 337 N1: 3.36(3H, s), 4.56(2H, br s), 8.15(1H, d, J=9.8 Hz) 66

M: 215-216 67

M: 270-271 N1: 3.74(4H, t, J=6.1 Hz), 7.40(1H, d, J=10.2 Hz), 9.47(1H,s) 68

M: 275-277 69

N1: 3.76(4H, t, J=6.1 Hz), 7.34(1H, d, J=10.4 Hz), 8.70(1H, s) 70

M: 98-99

TABLE 8 71

M: 202-203 72

M: 175-176 N1: 2.28(2H, br s), 3.86(3H, s), 5.84(1H, d, J=10.3 Hz) 73

M: 181-182 74

M: 152-153 75

M: 157-158 N1: 1.82(4H, br s), 8.12(1H, d, J=10.2 Hz), 8.78(1H, s) 76

M: 171-172 N1: 2.28(2H, br s), 5.84(1H, d, J=10.3 Hz), 8.64(1H, s) 77

N1: 3.60(4H, t, J=6.1 Hz), 4.79(2H, s), 7.15(1H, d, J=10.2 Hz) 78

M: 174-175 79

F: 354 N1: 3.71(1H, s), 5.41(2H, s), 8.05(1H, d, J=10.2 Hz) 80

M: 187-189 81

M: 126-127 82

N1: 3.88(3H, s), 7.43(1H, d, J=10.2 Hz), 10.74(1H, br s) 83

M: 116-117 84

M: 161-163 N1: 1.82(4H, br s), 7.28(1H, d, J=10.2 Hz), 8.42(1H, d, J=7.8Hz)

TABLE 9 85

F: 328 N1: 1.65(4H, br s), 3.95(3H, s), 8.12(1H, d, J=10.3 Hz) 86

F: 328 N1: 3.81(3H, s), 7.51(1H, dd, J=5.4, 3.0 Hz), 8.13(1H, d, J=10.3Hz) 87

M: 207-210 N1: 4.56(2H, br s), 7.33(1H, d, J=10.1 Hz), 8.42(1H, dd,J=7.8, 1.0 Hz) 88

N1: 3.98(3H, s), 4.89(1H, d, J=4.4 Hz), 8.15(1H, d, J=10.3 Hz) 89

M: 168-171 N1: 2.62(3H, s), 4.55(2H, br s), 7.33(1H, d, J=10.2 Hz) 90

F: 351 N1: 3.79(4H, d, J=4.0 Hz), 3.97(3H, s), 8.12(1H, d, J=10.2 Hz) 91

M: 108-110 92

M: 151-153 N1: 4.86(1H, dm J=46.9 Hz), 3.98(3H, s), 7.49(1H, d, J=10.3Hz) 93

M: 154-157 N1: 3.97(3H, s), 4.93(1H, dm, J=48.4 Hz), 7.50(1H, d, J=10.3Hz) 94

F: 337 N1: 3.04(2H, d, J=11.8 Hz), 3.97(3H, s), 8.13(1H, d, J=10.3 Hz)95

M: 178-180 N1: 2.06-2.16(4H, m), 3.97(3H, s,), 8.22(1H, d, 10.4 Hz) 96

M: 199-202 N1: 2.06-2.16(2H, m), 3.99(3H, s), 7.56(1H, d, J=10.3 Hz) 97

N1: 3.96(3H, s), 4.30(2H, d, J=12.2 Hz), 8.15(1H, d, J=10.3 Hz)

TABLE 10 98

N1: 3.97(3H, s), 4.51 (2H, d, J=14.1 Hz), 8.24(1H, d, J=10.2 Hz) 99

N1: 3.04(2H, d, J=11.8 Hz), 3.97(3H, s), 8.13(1H, d, J=10.3 Hz) 100

HCl M: 167-168 N1: 3.85(3H, s), 4.58(2H, m), 8.01(1H, d, J=7.8 Hz) 101

HCl M: 214-216(d) 102

HCl N1: 2.84(3H, s), 3.70(4H, t, J=5.9 Hz), 7.29(1H, d, J=10.4 Hz) 103

HCl M: 104-106 N1: 3.12(2H, t, J=7.2 Hz), 3.68(3H, s), 7.66(1H, d,J=10.4 Hz) 104

2HCl N1: 3.99(3H, s), 4.34(2H, d, J=13.2 Hz), 7.59(1H, d, J=10.2 Hz) 105

HCl N1: 4.00(3H, s), 7.02(1H, d, J=7.8 Hz), 8.19(1H, d, J=10.3 Hz) 106

HCl N1: 1.17(3H, t, J=7.1 Hz), 4.01(3H, s), 8.29(1H, d, J=10.2 Hz) 107

HCl F: 351 N1: 2.07(1H, br s), 4.01(3H, s), 8.24(1H, d, J=10.3 Hz) 108

HCl M: 133-135 109

M: 226-227 110

M: 178-179 111

M: 175-177

TABLE 11 112

M:124-125 N1:1.77(4H, br s), 2.62(3H, s), 7.31(1H, d, J=10.3Hz) 113

F:337 N1:3.45(3H, s), 4.00(3H, s), 8.03(1H, d, J=10.3Hz) 114

M:139-141 N1:7.73-1.84(4H, m), 2.62(3H, s), 7.28(1H, d, J=10.3Hz) 115

M:189-191 116

M:176-177 N1:1.63(6H, br s), 7.48(1H, d, J=10.2Hz), 7.76(1H, d, J=7.8Hz)117

M:169-171 N1:1.80(4H, br s), 7.31(1H, d, J=10.3Hz), 8.47(1H, d, J=7.6Hz)118

F:384 N1:4.57(2H, br s), 7.35(1H, d, J=10.0Hz), 8.49(1H, d, J=7.8Hz) 119

F:364 N1:1.42(3H, t, J=6.9Hz), 7.53(1H, d, J=16.6Hz), 8.06(1H, d,J=10.3Hz) 120

M:239-241 121

F:329 122

F:325 123

F:323

TABLE 12 No. RbRaN— —E—R 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

1. A nitrogen-containing heterocyclic compound represented by theformula (I):

or a pharmaceutically acceptable salt thereof, wherein Ra and Rb: thesame or different and each represents H; CO-lower alkyl; SO₂-loweralkyl; an optionally substituted cycloalkyl; an optionally substitutedaryl; or a lower alkyl which may have 1 to 3 substituents selected fromthe group consisting of an optionally substituted cycloalkyl, anoptionally substituted aryl, an optionally substituted 4- to 8-memberedmonocyclic saturated or partially unsaturated heterocyclic ring,CO-lower alkyl, SO₂-lower alkyl, OR¹, SR¹, NR¹R², halogen, NO₂, CN andCOOR¹; provided that at least one of Ra and Rb represents a group otherthan H; or, Ra and Rb taken together with an adjacent N atom represent a4- to 8-membered saturated or partially unsaturated heterocyclic ringcontaining 1 to 2 nitrogen atoms as heteroatoms, said heterocyclic ringmay be fused with a benzene ring or a cycloalkyl ring and may have abridge and may form a spiro ring, and said heterocyclic ring may have 1to 5 substituent, E: a single bond, C₁₋₃ alkylene, vinylene (—C═C—),ethynylene (—C≡C—), CO, NR³, CH₂-J, CONR⁴ or NR⁵CO, J: O, S, NR⁶, CO, SOor SO₂, R: an optionally substituted aryl, an optionally substitutedheteroaryl, an optionally substituted cycloalkyl, an optionallysubstituted cycloalkenyl or an optionally substituted 4- to 8-memberedmonocyclic saturated or partially saturated heterocyclic ring, R¹ to R⁶:the same or different and each denotes H or lower alkyl; with theproviso that the following compounds are excluded: (1) a compoundwherein Ra and Rb taken together with an adjacent N atom represent apiperidino, E is a single bond and R is a piperidino, unsubstitutedphenyl, p-(trifluoromethyl)phenyl, p-chlorophenyl or o-nitrophenyl, (2)a compound wherein Ra and Rb taken together with an adjacent N atomrepresent a 4-methyl-1-piperazinyl, E is a single bond, and R is anunsubstituted phenyl, p-methylphenyl, m-methylphenyl, p-methoxyphenyl,m-chlorophenyl, p-chlorophenyl or m-nitrophenyl, (3) a compound whereinR is an optionally substituted imidazolyl, 5-nitro-2-furyl or5-nitro-2-thienyl, (4) a compound wherein Ra is H, Rb is cyclopropyl, Eis a single bond and R is a p-(trifluoromethyl)phenyl, and, (5) acompound wherein Ra is a methyl, Rb is a 2-hydroxy-propyl, E is a singlebond and R is a 3-pyridyl.
 2. A nitrogen-containing heterocycliccompound according to claim 1 or a pharmaceutically acceptable saltthereof, wherein —NRaRb forms a 4- to 8-membered saturated or partiallyunsaturated heterocyclic ring having 1 to 2 N atoms as heteroatoms,which may be fused with a benzene ring or a cycloalkyl ring, which mayhave a bridge, or which may form a spiro ring, which may have 1 to 5substituents selected from Group B; wherein Group B comprises a loweralkyl which may have 1 to 4 substituents selected from Group G, halogen,NR¹R², —NR¹CO-lower alkyl, NO₂, CN, OR¹, —O-(lower alkyl having 1 to 4substituents selected from Group G), SR¹, —S-halogeno-lower alkyl,—O—CO-lower alkyl, COOR¹, COR¹, CONR¹R², SO-lower alkyl, SO₂-loweralkyl, SO₂NR¹R², P(═O) (OR¹)₂, —O—CH₂—O—, —O—(CH₂)₂—O—, aryl which mayhave 1 to 4 substituents selected from Group D, heteroaryl which mayhave 1 to 4 substituents selected from Group D, —O-(aryl which may have1 to 4 substituents selected from Group D), 4- to 8-membered monocyclicsaturated or partially unsaturated heterocyclic ring which may have 1 to4 substituents selected from Group D, cycloalkyl and —O-cycloalkyl;Group D comprises a lower alkyl, halogen, halogeno-lower alkyl, NR¹R²,NO₂, CN, OR¹ and SR¹; Group G comprises a halogen, NR¹R², CN, COOR¹,OR¹, SR¹, 4- to 8-membered monocyclic saturated or partially unsaturatedheterocyclic ring which may have 1 to 4 substituents selected from GroupD, aryl which may have 1 to 4 substituents selected from Group D andheteroaryl which may have 1 to 4 substituents selected from Group D; Eis a single bond, C₁₋₃ alkylene, vinylene, ethynylene, CONH, CH₂NH, CH₂Oor CH₂S; R is an aryl which may have 1 to 5 substituents selected fromGroup B or heteroaryl which may have 1 to 5 substituents selected fromGroup B.
 3. A nitrogen-containing heterocyclic compound according toclaim 2 or a pharmaceutically acceptable salt thereof, wherein E is asingle bond, C₁₋₃ alkylene, vinylene or ethynylene; R is an aryl having1 to 5 substituents selected from Group B1 or heteroaryl having 1 to 5substituents selected from Group B1; Group B1 comprises a lower alkyl,halogeno-lower alkyl, halogen, NR¹R², NO₂, CN, OR¹, —O-halogeno-loweralkyl, SR¹, COOR¹, CONR¹R², SO₂-lower alkyl, 4- to 8-membered monocyclicsaturated or partially unsaturated heterocyclic ring, phenyl andphenoxy.
 4. A nitrogen-containing heterocyclic compound according toclaim 3 or a pharmaceutically acceptable salt thereof, wherein —NRaRb isa 4- to 8-membered monocyclic saturated or partially unsaturatedheterocyclic ring which may have one N atom as a heteroatom and may havea bridge, and may have 1 to 2 substituents selected from a lower alkyl,halogen, OR¹ and COOR¹; E is a single bond; R is a phenyl having asubstituent in its m-position selected from a halogen, O-lower alkyl andS-lower alkyl or a pyridyl having a substituent in its 6-positionselected from a halogen, O-lower alkyl and S-lower alkyl.
 5. Anitrogen-containing heterocyclic compound according to claim 1 or apharmaceutically acceptable salt thereof selected from6-azocan-1-yl-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azepan-1-yl-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,3-(3-methoxyphenyl)-6-(piperidin-1-yl)-1,2,14-triazolo[4,3-b]pyridazine,3-(3-bromophenyl)-6-(piperidin-1-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azepan-1-yl-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(4-fluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(3-azabicyclo[3.2.1]octan-3-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(4,4-difluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-(3,3-difluoropiperidin-1-yl)-3-(6-methoxypyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,6-azocan-1-yl-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine,and6-(8-azabicyclo[3.2.1]octan-8-yl)-3-(6-bromopyridin-2-yl)-1,2,4-triazolo[4,3-b]pyridazine.6. A pharmaceutical composition comprising a nitrogen-containingheterocyclic compound according to claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 7. Apharmaceutical composition according to claim 6 which is a bone-formingagent.
 8. Use of a nitrogen-containing heterocyclic compound accordingto claim 1 or a pharmaceutically acceptable salt thereof for themanufacture of a bone-forming agent for a mammalian animal.
 9. A methodfor stimulating bone formation in a mammalian animal, comprisingadministering an effective amount of a nitrogen-containing heterocycliccompound according to claim 1 or a pharmaceutically acceptable saltthereof to the mammalian animal.